Assembly of the Light-Harvesting Complexes (LHCs) of Photosystem II (Monomeric LHC IIb Complexes Are Intermediates in the Formation of Oligomeric LHC IIb Complexes)

The light-induced assembly of light-harvesting complex (LHC) II has been followed during the biogenesis of the plastid. Seedlings grown in intermittent light (IML) accumulate only small amounts of chlorophyll b. The minor LHC II apoproteins are present; however, the apoprotein levels of the major LHC II complex, LHC IIb, are severely depressed after exposure to IML. The levels of all LHC II apoproteins increase rapidly upon exposure to continuous illumination. The 25-kD, type 3 LHC IIb subunit appears to be more abundant during the early hours of greening in relation to its level in mature thylakoids. The LHC IIb apoproteins are initially associated with pigments to form monomeric pigment-protein complexes. The abundance of monomeric LHC IIb complexes gradually decreases during exposure to continuous light and a concomitant increase occurs in the amount of the trimeric and higher-order oligomeric forms. Pulse-chase experiments verify that labeled LHC IIb monomeric complexes are intermediates in the formation of trimeric and higher-order oligomeric LHC IIb-pigmented complexes. Therefore, the assembly of LHC II occurs via the initial pigmentation of the apoproteins to form monomeric complexes and proceeds in a sequential manner.     

Protein domains required for formation of stable monomeric Lhca1- and Lhca4-complexes

The peripheral light-harvesting complex of Photosystem I consists of two subpopulations, LHC I-680 and LHC I-730. The latter is composed of the two apoproteins Lhca1 and Lhca4. Recently, reconstitution of monomeric LHC I using bacterially overexpressed Lhca1 or Lhca4 was achieved. In order to obtain insight into the structure requirements for formation of monomeric light-harvesting complexes, we produced a series of N- and C-terminal deletion mutants and used the overexpressed proteins for reconstitution experiments. We found the entire extrinsic N-terminal region dispensable for monomer formation in Lhca1 and Lhca4. Also at the C-terminus, both subunits revealed similarity since all amino acids up to the end of the fourth helix could be removed without abolishing monomer formation. In connection with former corresponding results for Lhcb1, the dispensability of these regions appears to be a general feature in LHC-formation. In LHC I, however, a stabilising effect can be ascribed to these regions since the yield of complexes was decreased. In the majority of the mutant LHC I versions no effect on pigment binding was detected. However, in the LHC with the most extensively N-terminally truncated mutant of Lhca4 a dramatic shift in the 77 K fluorescence emission to shorter wavelengths was observed. This suggests that chlorophylls involved in long wavelength fluorescence emission are located in the chlorophyll array located towards the stromal face of the thylakoid membrane assuming a pigment arrangement corresponding to that in LHC II and CP29. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of the subunit compositions of the PSI-LHCI supercomplex and the LHCI in the green alga Chlamydomonas reinhardtii

Although the light-harvesting chlorophyll protein complex I (LHCI) of photosystem I (PSI) is intimately associated with the PSI core complex and forms the PSI-LHCI supercomplex, the LHCI is normally synthesized in PSI-deficient mutants. In this paper, we compared the subunit compositions of the PSI-LHCI supercomplex and the LHCI by immunoblot analysis and two-dimensional gel electrophoresis combined with mass spectrometry. The PSI-LHCI supercomplex and the LHCI were purified by sucrose density gradient centrifugation and (diethylamino)ethyl column chromatography from n-dodecyl-beta-D-maltoside-solubilized thylakoids of the wild-type and DeltapsaB mutant of the green alga Chlamydomonas reinhardtii. The PSI-LHCI supercomplex contained all of the nine Lhca polypeptides (Lhca1-9) that are detected in wild-type thylakoids. In contrast, the LHCI retained only six Lhca polypeptides, whereas Lhca3 and two minor polypeptides, Lhca2 and Lhca9, were lost during the purification procedure. Sucrose density gradient centrifugation showed that the purified LHCI retains an oligomeric structure with an apparent molecular mass of 300-400 kDa. We therefore concluded that Lhca2, Lhca3, and Lhca9 are not required for the stable oligomeric structure of the LHCI and that the association of these polypeptides in the LHCI is stabilized by the presence of the PSI core complex. Finally, we discuss the possible localization and function of Lhca polypeptides in the LHCI.     

Lhca5 – an LHC-Type Protein Associated with Photosystem I

The light-harvesting antenna of higher plant photosystem (PS) I is known to be composed of four different types of light-harvesting complex (LHC) proteins (Lhca1-4). However, the genomic sequence of Arabidopsis thaliana contains open reading frames coding for two additional LHC type proteins (Lhca5-6) that are presumably associated with PSI. While Lhca6 might not be expressed at all, ESTs have been detected for the Lhca5 gene in Arabidopsis and a number of other plant species. Here we demonstrate the presence of the Lhca5 gene product in the thylakoid membrane of Arabidopsis as an additional type of Lhca-protein associated with PSI. Lhca5 seems to be regulated differently from the other LHC proteins since Lhca5 mRNA levels increase under high light conditions. Analyses reported here of Lhca5 in plants lacking individual Lhca1-4 proteins show that it is more abundant in plants lacking Lhca1/4, and suggest that it interacts in a direct physical fashion with Lhca2 or Lhca3. We propose that Lhca5 binds chlorophylls in a similar fashion to the other Lhca proteins and is associated with PSI only in sub-stoichiometric amounts.     

Light-harvesting pigment-proteins of photosystem I in maize. Subunit composition and biogenesis

Three different pigment-binding proteins of the light-harvesting complex (LHC I) of maize photosystem I (PS I) have been isolated. Absorption and fluorescence excitation spectral analyses showed that each pigment-protein can transfer absorbed energy from its carotenoid and/or chlorophyll b components to chlorophyll alpha. Their apoproteins with apparent sizes of 24 (LHC Ia), 21 (LHC Ib), and 17 (LHC Ic) kDa have been purified to homogeneity. Differences in their pigment and amino acid compositions and in their reactions with antibodies demonstrate that the two smaller pigment-proteins are not proteolytically derived from the largest one. LHC Ib's apoprotein is particularly enriched in cysteine residues. None of the three apoproteins cross-reacted with antibodies raised against the major light-harvesting chlorophyll a/b-protein of photosystem II (LHC IIb) or against the PS I core complex (CC I) subunits. Studies of the biogenesis of PS I during greening of etiolated plants showed that all of the CC I subunits accumulated to a detectable level prior to the appearance of the 17-kDa subunit of LHC I, the accumulation of which preceded those of the 24- and 21-kDa subunits of LHC I. In addition, subunit VI of CC I is shown to be differentially expressed in mesophyll and bundle sheath cells; a slightly larger form of it accumulates in mesophyll than in bundle sheath thylakoids during plastid development.     

Analysis of the pigment stoichiometry of pigment-protein complexes from barley (Hordeum vulgare). The xanthophyll cycle intermediates occur mainly in the light-harvesting complexes of photosystem I and photosystem II.

The carotenoid zeaxanthin has been implicated in a nonradiative dissipation of excess excitation energy. To determine its site of action, we have examined the location of zeaxanthin within the thylakoid membrane components. Five pigment-protein complexes were isolated with little loss of pigments: photosystem I (PSI); core complex (CC) I, the core of PSI; CC II, the core of photosystem II (PSII); light-harvesting complex (LHC) IIb, a trimer of the major light-harvesting protein of PSII; and LHC IIa, c, and d, a complex of the monomeric minor light-harvesting proteins of PSII. Zeaxanthin was found predominantly in the LHC complexes. Lesser amounts were present in the CCs possibly because these contained some extraneous LHC polypeptides. The LHC IIb trimer and the monomeric LHC II a, c, and d pigment-proteins from dark-adapted plants each contained, in addition to lutein and neoxanthin, one violaxanthin molecule but little antheraxanthin and no zeaxanthin. Following illumination, each complex had a reduced violaxanthin content, but now more antheraxanthin and zeaxanthin were present. PSI had little or no neoxanthin. The pigment content of LHC I was deduced by subtracting the pigment content of CC I from that of PSI. Our best estimate for the carotenoid content of a LHC IIb trimer from dark-adapted plants is one violaxanthin, two neoxanthins, six luteins, and 0.03 mol of antheraxanthin per mol trimer. The xanthophyll cycle occurs mainly or exclusively within the light-harvesting antennae of both photosystems.     

Protein-protein interactions by molecular modeling and biochemical characterization of PSI-LHCI supercomplexes from Chlamydomonas reinhardtii

The physiological function of Photosystem I (PSI) is a sunlight energy converter, catalyzing one of the initial steps in driving oxygenic photosynthesis in cyanobacteria, algae and higher plants. The Chlamydomonas reinhardtii PSI structure was not known since it contains a unique structure having additional light harvesting complex I (LHCI) subunits, which play a major role in the transfer of sunlight energy to the reaction center. Here, individual subunits of LHC and core subunits are built based on the PDB taken from RCSB Protein Data Bank. The model gives information about the geometrical existence of subunits following a flanking order of Lhca5, Lhca1, Lhca6, Lhca4, Lhca2, Lhca8, Lhca9, Lhca7, and Lhca3. The new subunit PsaO is located close to the PsaH, PsaI and PsaL subunits, thus it may be involved in the state transition mechanism and stabilization of PSI-LHCI supercomplexes. The modeled PSI-LHCI structure of C. reinhardtii shows a unique arrangement of PsaN, PsaO of PSI core subunits and Lhca5 to Lhca9 of LHCI subunits. There are many non-covalent interactions among the PSI and LHCI subunits, which suggest that C. reinhardtii PSI-LHCI supercomplexes are more complex than higher plants. These results strongly support the experimental data that, even with harsh treatment of the PSI-LHCI supercomplexes with detergent, the complexes do not dissociate due to strong interactions between the PSI core and LHCI. Thus, our 3D model may give valid structural information of the PSI-LHCI arrangement and its physiological role in C. reinhardtii.     

Light-Induced Biogenesis of Chlorophyll–Protein Complexes in Developing Wheat Thylakoids

It is shown that the apoproteins of core complexes (CC) I and II, the - and -subunits of CF₁ ATP-synthase complexes, are present in seedlings grown under intermittent light (IML). The levels of light-harvesting complex (LHC) apoproteins in the 30- to 18-kD region increase rapidly upon exposure to continuous light (CL). The newly synthesized LHC apoproteins appear to be present predominantly in monomeric forms that later assemble into higher-order oligomeric forms. During the early stages of greening of wheat seedlings, polypeptides in the 20.5-19 and 17.5-15.5 kD regions (so-called early light-induced proteins (ELIP)) are observed, but they disappear fully after 6 h. As greening proceeds, the 727-nm band in low-temperature fluorescence spectra (77 K) gradually shifts to longer wavelength (740-742 nm), which clearly demonstrates the light-driven biogenesis of LHC I and its assembly with CC I.     

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri

Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment-protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715-730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.     

Excitation-energy transfer dynamics of higher plant photosystem I light-harvesting complexes

Photosystem I (PSI) plays a major role in the light reactions of photosynthesis. In higher plants, PSI is composed of a core complex and four outer antennas that are assembled as two dimers, Lhca1/4 and Lhca2/3. Time-resolved fluorescence measurements on the isolated dimers show very similar kinetics. The intermonomer transfer processes are resolved using target analysis. They occur at rates similar to those observed in transfer to the PSI core, suggesting competition between the two transfer pathways. It appears that each dimer is adopting various conformations that correspond to different lifetimes and emission spectra. A special feature of the Lhca complexes is the presence of an absorption band at low energy, originating from an excitonic state of a chlorophyll dimer, mixed with a charge-transfer state. These low-energy bands have high oscillator strengths and they are superradiant in both Lhca1/4 and Lhca2/3. This challenges the view that the low-energy charge-transfer state always functions as a quencher in plant Lhc's and it also challenges previous interpretations of PSI kinetics. The very similar properties of the low-energy states of both dimers indicate that the organization of the involved chlorophylls should also be similar, in disagreement with the available structural data.     

Isolation and characterization of a large photosystem I–light‐harvesting complex II supercomplex with an additional Lhca1–a4 dimer in Arabidopsis

The biological conversion of light energy into chemical energy is performed by a flexible photosynthetic machinery located in the thylakoid membranes. Photosystems I and II (PSI and PSII) are the two complexes able to harvest light. PSI is the last complex of the electron transport chain and is composed of multiple subunits: the proteins building the catalytic core complex that are well conserved between oxygenic photosynthetic organisms, and, in green organisms, the membrane light‐harvesting complexes (Lhc) necessary to increase light absorption. In plants, four Lhca proteins (Lhca1–4) make up the antenna system of PSI, which can be further extended to optimize photosynthesis by reversible binding of LHCII, the main antenna complex of photosystem II. Here, we used biochemistry and electron microscopy in Arabidopsis to reveal a previously unknown supercomplex of PSI with LHCII that contains an additional Lhca1–a4 dimer bound on the PsaB–PsaI–PsaH side of the complex. This finding contradicts recent structural studies suggesting that the presence of an Lhca dimer at this position is an exclusive feature of algal PSI. We discuss the features of the additional Lhca dimer in the large plant PSI–LHCII supercomplex and the differences with the algal PSI. Our work provides further insights into the intricate structural plasticity of photosystems.     

The oligomeric states of the photosystems and the light-harvesting complexes in the Chl b-less mutant

The reversible associations between the light-harvesting complexes (LHCs) and the core complexes of PSI and PSII are essential for the photoacclimation mechanisms in higher plants. Two types of Chls, Chl a and Chl b, both function in light harvesting and are required for the biogenesis of the photosystems. Chl b-less plants have been studied to determine the function of the LHCs because the Chl b deficiency has severe effects specific to the LHCs. Previous studies have shown that the amounts of the LHCs, especially the LHCII trimer, were decreased in the mutants; however, it is still unclear whether Chl b is required for the assembly of the LHCs and for the association of the LHCs with PSI and PSII. Here, to reveal the function of Chl b in the LHCs, we investigated the oligomeric states of the LHCs, PSI and PSII in the Arabidopsis Chl b-less mutant. A two-dimensional blue native-PAGE/SDS-PAGE demonstrated that the PSI-LHCI supercomplex was fully assembled in the absence of Chl b, whereas the trimeric LHCII and PSII-LHCII supercomplexes were not detected. The PSI-NAD(P)H dehydrogenase (NDH) supercomplexes were also assembled in the mutant. Furthermore, we detected two forms of monomeric LHC proteins. The faster migrating forms, which were detected primarily in the mutant, were probably apo-LHC proteins, whereas the slower migrating forms were probably the LHC proteins that contained Chl a. These findings increase our understanding of the Chl b function in the assembly of LHCs and the association of the LHCs with PSI, PSII and NDH.     

Light-Harvesting Features Revealed by the Structure of Plant Photosystem I

Oxygenic photosynthesis is driven by two multi-subunit membrane protein complexes, Photosystem I and Photosystem II. In plants and green algae, both complexes are composed of two moieties: a reaction center (RC), where light-induced charge translocation occurs, and a peripheral antenna that absorbs light and funnels its energy to the reaction center. The peripheral antenna of PS I (LHC I) is composed of four gene products (Lhca 1-4) that are unique among the chlorophyll a/b binding proteins in their pronounced long-wavelength absorbance and in their assembly into dimers. The recently determined structure of plant Photosystem I provides the first relatively high-resolution structural model of a super-complex containing a reaction center and its peripheral antenna. We describe some of the structural features responsible for the unique properties of LHC I and discuss the advantages of the particular LHC I dimerization mode over monomeric or trimeric forms. In addition, we delineate some of the interactions between the peripheral antenna and the reaction center and discuss how they serve the purpose of dynamically altering the composition of LHC I in response to environmental pressure. Combining structural insight with spectroscopic data, we propose how altering LHC I composition may protect PS I from excessive light.     

Protective effect of active oxygen scavengers on protein degradation and photochemical function in photosystem I submembrane fractions during light stress

The protective role of reactive oxygen scavengers against photodamage was studied in isolated photosystem (PS) I submembrane fractions illuminated (2000 microE x m(-2) x s(-1)) for various periods at 4 degrees C. The photochemical activity of the submembrane fractions measured as P700 photooxidation was significantly protected in the presence of histidine or n-propyl gallate. Chlorophyll photobleaching resulting in a decrease of absorbance and fluorescence, and a blue-shift of both absorbance and fluorescence maximum in the red region, was also greatly delayed in the presence of these scavengers. Western blot analysis revealed the light harvesting antenna complexes of PSI, Lhca2 and Lhca1, were more susceptible to strong light when compared to Lhca3 and Lhca4. The reaction-center proteins PsaB, PsaC, and PsaE were most sensitive to strong illumination while other polypeptides were less affected. Addition of histidine or n-propyl gallate lead to significant protection of reaction-center proteins as well as Lhca against strong illumination. Circular dichroism (CD) spectra revealed that the alpha-helix content decreased with increasing period of light exposure, whereas beta-strands, turns, and unordered structure increased. This unfolding was prevented with the addition of histidine or n-propyl gallate even after 10 h of strong illumination. Catalase or superoxide dismutase could not minimize the alteration of PSI photochemical activity and structure due to photodamage. The specific action of histidine and n-propyl gallate indicates that 1O2 was the main form of reactive oxygen species responsible for strong light-induced damage in PSI submembrane fractions.     

Pigment binding of photosystem I light-harvesting proteins

Light-harvesting complexes (LHC) of higher plants are composed of at least 10 different proteins. Despite their pronounced amino acid sequence homology, the LHC of photosystem II show differences in pigment binding that are interpreted in terms of partly different functions. By contrast, there is only scarce knowledge about the pigment composition of LHC of photosystem I, and consequently no concept of potentially different functions of the various LHCI exists. For better insight into this issue, we isolated native LHCI-730 and LHCI-680. Pigment analyses revealed that LHCI-730 binds more chlorophyll and violaxanthin than LHCI-680. For the first time all LHCI complexes are now available in their recombinant form; their analysis allowed further dissection of pigment binding by individual LHCI proteins and analysis of pigment requirements for LHCI formation. By these different approaches a correlation between the requirement of a single chlorophyll species for LHC formation and the chlorophyll a/b ratio of LHCs could be detected, and indications regarding occupation of carotenoid-binding sites were obtained. Additionally the reconstitution approach allowed assignment of spectral features observed in native LHCI-680 to its components Lhca2 and Lhca3. It is suggested that excitation energy migrates from chlorophyll(s) fluorescing at 680 (Lhca3) via those fluorescing at 686/702 nm (Lhca2) or 720 nm (Lhca3) to the photosystem I core chlorophylls.     

Structural characterization of a plant photosystem I and NAD(P)H dehydrogenase supercomplex

Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI–NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo‐atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low‐abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI–NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.     

Differential degradation of photosystem I subunits under iron deficiency in rice

Rice (Oryza sativa) is one of the staple foods of the world. Iron (Fe) deficiency is a major abiotic stress factor that contributes world-wide to losses in crop yield and decline in nutritional quality. As cofactor for many enzymes and proteins, iron is an essential element. It plays a pivotal role in chlorophyll (Chl) biosynthesis, and iron deficiency may result in decreased Chl production and, thus, reduced photosynthetic capacity. Photosystem I (PSI) is a prime target of iron deficiency because of its high iron content (12 Fe per PS). To understand the protein level changes in the light-harvesting complex (LHC) of PSI (LHCI) under iron deficiency, rice seedlings were grown in Hoagland's nutrient medium with and without Fe. Chlorophyll content and photosynthetic efficiency decreased under iron deficiency. Protein gel blots probed with antibodies against the PSI core and Lhca 1-4 proteins revealed that the core subunits PsaA and PsaB remained stable under iron deficiency, whereas PsaC and PsaD decreased by about 50%, and PsaE was completely degraded. Among the LHCI subunits, Lhca1 and Lhca2 decreased by 40 and 50%, respectively, whereas Lhca3 and Lhca4 were completely degraded. We propose that the dissociation of LHCI subunits may be due to increased levels of reactive oxygen species, which is suggested by the increased activity of superoxide dismutase.     

Rapid isolation of photosystem I chlorophyll-binding proteins by anion exchange perfusion chromatography

With the new method of anion exchange perfusion chromatography we have devised an extremely rapid technique to subfractionate spinach Photosystem I into its chlorophyll a containing core complex and various components of the Photosystem I light-harvesting antenna (LHC I). The isolation time for the LHC I subcomplexes following solubilisation of native Photosystem I was reduced from 50 h using traditional density centrifugation procedures down to only 10–25 min by perfusion chromatography. Within this very short period of isolation, LHC I has been obtained as subfractions highly enriched in Lhca2+3 (LHC I-680) and Lhca1+4 (LHC I-730). Moreover, other highly enriched subfractions of LHC I such as Lhca2, Lhca3 and Lhca1+2+4 were obtained where the later two populations have not previously been obtained in a soluble form and without the use of SDS. These various subfractions of the LHC I antenna have been characterised by absorption spectroscopy, 77 K fluorescence-spectroscopy and SDS-PAGE demonstrating their identities, functional intactness and purity. Furthermore, the analyses located a chlorophyll b pool to preferentially transfer its excitation energy to the low energy F735 chromophore, and located specifically the origin of the 730 nm fluorescence to the Lhca4 component. It was also revealed that Lhca2 and Lhca3 have identical light-harvesting properties. The isolated Photosystem I core complex showed high electron transport capacity (1535 moles O₂ mg Chl^–1 h^–1) and low fluorescence yield (0.4%) demonstrating its high functional integrity. The very rapid isolation procedure based upon perfusion chromatography should in a significant way facilitate the subfractionation of Photosystem I proteins and thereby allow more accurate functional and structural studies of individual components.Abbreviations a.u. arbitrary units - DCIP 2.6-dichlorophenol indophenol - LHC light harvesting complex     

Specific substitutions of light‐harvesting complex I proteins associated with photosystem I are required for supercomplex formation with chloroplast NADH dehydrogenase‐like complex

In Arabidopsis, the chloroplast NADH‐dehydrogenase‐like (NDH) complex is sandwiched between two copies of photosystem I (PSI) supercomplex, consisting of a PSI core and four light‐harvesting complex I (LHCI) proteins (PSI‐LHCI) to form the NDH–PSI supercomplex. Two minor LHCI proteins, Lhca5 and Lhca6, contribute to the interaction of each PSI–LHCI copy with the NDH complex. Here, large‐pore blue‐native gel electrophoresis revealed that, in addition to this complex, there were at least two types of higher‐order association of more LHCI copies with the NDH complex. In single‐particle images, this higher‐order association of PSI–LHCI preferentially occurs at the left side of the NDH complex when viewed from the stromal side, placing subcomplex A at the top (Yadav et al., Biochim. Biophys. Acta ‐ Bioenerg., 1858, 2017, 12). The association was impaired in the lhca6 mutant but not in the lhca5 mutant, suggesting that the left copy of PSI–LHCI was linked to the NDH complex via Lhca6. From an analysis of subunit compositions of the NDH–PSI supercomplex in lhca5 and lhca6 mutants, we propose that Lhca6 substitutes for Lhca2 in the left copy of PSI–LHCI, whereas Lhca5 substitutes for Lhca4 in the right copy. In the lhca2 mutant, Lhca3 was specifically stabilized in the NDH–PSI supercomplex through heterodimer formation with Lhca6. In the left copy of PSI–LHCI, subcomplex B, Lhca6 and NdhD likely formed the core of the supercomplex interaction. In contrast, a larger protein complex, including at least subcomplexes B and L and NdhB, was needed to form the contact site with Lhca5 in the right copy of PSI–LHCI.